Project Summary Prolonged use opioids results in a paradoxical increase in atypical pain (hyperalgesia). Despite its known limitations, mechanisms underlying opioid-induced hyperalgesia still remains a significant gap in knowledge. Peripheral neuropathy has become the most common neurologic complication in HIV patients, with prevalence as high as 69.4% in infected patients, Notably, HIV-infected opioid abusers appear to exhibit more severe neuropathy than HIV-infected non-drug users. Furthermore, repeated use of opioid analgesics are reported to promote neuropathic pain in HIV patients. Very little is known if ART contributes to peripheral neuropathy and if it is exacerbated in chronic opioid users. The main metabolic pathway for morphine and morphine derivatives in mammals is glucuronidation catalyzed by UDP-glucuronosyltransferase (UGT) enzymes. Morphine is metabolized predominantly to two metabolites, morphine-3-glucuronide (M3G) and morphine-6-glucuronide (M6G) (1,2). M-3-G is the only metabolite produced in rodents and does not have any analgesic effects but have been shown in recent studies to activate and modulate TLR signaling. Furthermore, antiretroviral drugs particularly, intergrase inhibitors, are also metabolized through the UGT- glucuronidation pathway. Recent studies show that the metabolites of the intergrase inhibitor zidovudine to be more toxic than the parent compound. In preliminary data we show that chronic morphine treatment results in significant gut microbial dysbiosis with significant decrease in bacterial communities that synthesize ?-glucuronidase with a concurrent decrease in glucuronidase activity. We show in preliminary data that depletion in the bacterial communities involved in the synthesis of ? -glucuronidase results in a) increased accumulation M-3-G in the gut and serum and b) reduced enterohepatic recycling of morphine. We further demonstrate that morphine treatment in the context of HIV infection results in significant hyperalgesia in the both the Mechanical allodynia (Von Frey) and Thermal hyperalgesia test (Hargraves) tests. Proinflammatory cytokine levels and morphine induced hyperalgesia are significantly attenuated in TLR2 knock out mice. Based on these observations we hypothesize that decreased microbial communities associated with ? glucuronidase synthesis following prolonged exposure to high dose morphine results in accumulation of M-3-G morphine metabolite and toxic glucuronidated ART in the small intestine contributing to Specific Aim 1: Establish that accumulation of the morphine metabolite M-3-glucoronide in the gut results in opioid induced hyperalgesia and HIV induced peripheral neuropathy in opiod using HIV patients. Specific Aim 2: Determine the role of TLR2 signaling in morphine induced hyperalgesia and HIV associated peripheral neuropathy in the context of ART treatment. Specific Aim 3: Treatment with genetically engineered commensal bacteria with controlled expression of B- glucuronidase in combination with TLR2/4 antagonist will preserve small intestinal barrier integrity and attenuate peripheral neuropathy and prolong morphine analgesia.